The invention relates to the structure of sample support plates for the mass spectrometric analysis of samples with ionization by matrix-assisted laser desorption and ionization (MALDI).
Mass spectrometry with ionization by matrix-assisted laser desorption and ionization (MALDI) is now established as a standard method for the analysis of biomolecules. In most cases, time-of-flight mass spectrometers (TOF-MS) are used, but ion-cyclotron resonance spectrometers or radio-frequency quadrupole ion-trap mass spectrometers can also be applied.
The biomolecules are usually in aqueous solution. Here, biomolecules are understood in particular as oligonucleotides (i.e., genetic material in its different forms, such as DNA or RNA) and proteins (i.e., the essential building blocks of the living world), including their particular analogs and conjugates such as glycoprpteins or lipoproteins. Ionization by MALDI can also be used for industrial polymers and small organic compounds. In the following, the molecules being analyzed are referred to as sample molecules or analyte molecules.
The choice of matrix substance for the MALDI process depends on the type of biomolecules. Well over a hundred different matrix substances with their different merits are now known. In particular, the matrix substance must absorb light at the laser wavelength being used, but must also isolate the test molecules from each other in an appropriate manner, convert them into the gaseous phase intact (desorption) and ionize them (usually by protonation or deprotonation). For this task, it has been found to be advantageous to incorporate the analyte molecules in some form into the, in most cases, crystalline matrices as they crystallize on the surface of the sample support or at least into the boundary surfaces between the small crystals which form during the crystallization. There are 103 to 105 times as many matrix molecules as there are analyte molecules.
A range of different methods are known for laying down the sample and matrix. The simplest of these is to pipette a solution of the sample and matrix onto a clean metallic sample support. The drop of solution forms a wetted area on the metal surface. The diameter of the drop is determined by the wettability of the particular metal surface being used. As the solution dries, a sample spot forms which contains tiny matrix crystals within the wetted area. However, the coating on the wetted surface is usually not uniform. With many matrix substances, the tiny crystals are located at the edge of the sample spot. Here, so-called xe2x80x98hot spotsxe2x80x99 of high sensitivity form which cannot be recognized as such without testing.
For matrix substances which are either insoluble or only very sparingly soluble in water, such as xcex1-cyano-4-hydroxycinnamic acid, it has been found to be advantageous to produce a very thin layer of crystals on the surface before applying the aqueous analyte solutions, for example by applying a solution of the matrix substance in acetone. In this case, the sensitivity is more uniform over the coating area.
An improved method of laying down the sample is disclosed in the patent specification DE 197 54 978 C1 (GB 2 332 273, U.S. Pat. No. 6,287,872) which consists of applying the samples to small hydrophilic anchor zones in a hydrophobic field. Drops containing the dissolved matrix and dissolved analyte molecules which have been pipetted onto the surface attach themselves to the anchor zones where they crystallize much more uniformly than on surfaces without anchors. The crystalline conglomerates bond strongly to the hydrophilic anchor zones on the surface of the sample support. With careful preparation, it is possible to achieve a sensitivity which is both uniform and reproducible. Here too, it is possible to apply the matrix substances before applying the sample solutions.
All these methods of applying the samples and incorporating them into the tiny matrix crystals are highly dependent on the properties of the hydrophilic anchor zones. These properties include the chemical composition of the sample support at its surface, the oxidation state of the surface, the smoothness of the surface and, in particular, the wetting properties of the surface toward the solvent used. Of crucial importance is that the surface is extremely clean, since the MALDI process can be disturbed by even the tiniest trace of impurities. In particular, no alkali ions must pass from the surface into the dissolved sample. With the usually metallic surfaces of sample supports, reproducible surface structures with the specified properties can only be achieved with great difficulty.
If time-of-flight mass spectrometers are used for the analysis, then the sample supports must also be exceptionally flat. Any twist in the surface must not exceed a few microns otherwise the precise mass determination required to achieve today""s accuracies of a few ppm (parts per million) will be more difficult to obtain because of the differences in the length of the flight path. For a flight of one meter, lengthening the flight path by one micron corresponds to an increase in the time of flight of about a millionth and an apparent increase in the mass of two millionths.
So far, only a few types of sample support materials have been found to have a certain degree of universal application. These include, in particular, (1) smooth-rolled, three millimeter stainless steel sheet made by using a special annealing process and with a ground or polished surface, (2) glass plates coated with electrically conductive material, (3) aluminum plates coated with nickel or gold and (4) silicon wafer plates. Since the condition of the surface is of critical importance for the crystallization of the matrix, and different matrices are used according to the application, in practice, different sample support plates are preferred depending on the-application.
For the automated handling of sample support plates, it is advantageous for the plates to have the shape that has become the industry standard for microtitre plates. Commercially available pipette robots can only process sample support plates with the approximate shape of microtitre plates. The plates can be held by standardized grippers and populated with sample droplets using multi-pipette heads. They can be stacked in xe2x80x9cplate hotelsxe2x80x9d or inserted into appropriate magazines like a chest of drawers. The shape of the underside of the microtitre plates acts as a relatively tight, or at least dust-proof, seal for the plate underneath.
The sample support plates can be provided with bar codes on the front or on top. The bar code can be read by various industrial robots. However, it is difficult to develop a printed bar code to withstand a vacuum or washing. For this reason, sample support plates have been developed with vacuum- and wash-proof transponders with readable codes. In some cases, it is even possible to provide the transponder with the current status of the population along with other information.
The use of MALDI sample supports in the shape of microtitre plates for coating with samples from multi-pipetting heads has already been described in the patent specification DE 196 28 178 C2 (corresponding to GB 2 315 329, U.S. Pat. No. 5,770,860).
Many attempts have been made at making sample support plates for use in the MALDI process from plastic. There are a very large number of different plastics. They can be molded extremely cheaply and, with the appropriate fillers, can also be made electrically conductive. It is possible to produce the desired surface textures with a very high level of reproducibility. The surfaces can be metallized, made scratch resistant and made hydrophobic in many different ways. In short, there is hardly any other material with so many possibilities. However, plastics have one crucial disadvantage: they are not resistant to deformation and they go out of shape very easily after molding. Even storage changes their shape. A degree of evenness to within a few microns on larger surfaces is not easily achieved or does not remain stable over long periods.
Nevertheless, the cost-effective manufacture of MALDI sample support plates is still worth striving for. With reusable sample supports, the so-called memory effect can have a negative influence on the measurement results, particularly for applications requiring the highest sensitivity, and certainly for diagnostic applications, since in many cases the analyte molecules cannot be removed quantitatively even with careful washing. Apart from that, there is an increasing demand for test sites which are industrially pre-coated with tested matrix substances of guaranteed purity and function. But further steps in sample preparation on sample support plates, such as enzymic digestion, purification of the test substances or markings, are also approaching and require cheap, disposable sample support plates.
The basic idea of the invention is a composite sample support with a structure consisting of a reusable substructure made from a mechanically very stable material with the highest dimensional accuracy and a removable plastic cover. Preferredly, the plastic cover is to be used once only. The substructure can be made of materials such as stainless steel, with a surface which is even and dimensionally accurate enough to be used as a MALDI sample support plate. The disposable cover is a plate of uniform thickness made from a relatively thin plastic which is attached so that it lies flush on the substructure. Nowadays it is possible to manufacture plastic plates of uniform thickness and with tolerances of only a few microns on a large scale by injection moulding etc.
Flush mounting can be achieved in different ways. For example, a large number of tags on the plastic plate can be pressed into a large number of undercut holes or grooves in the substructure. However, it is seems better to produce a slightly concave plate with a smooth bottom surface which can be pressed firmly onto the substructure at the edge and will remain flush over a large area due to its elasticity. However, the plastic cover must not be pressed too far over the substructure. The plastic plate can be held at the edges by a separate frame fixed onto the substructure or by a retaining edge on the plastic plate in the form of a solid or perforated strip around the edge which is retained by appropriate ridges or grooves. The grooves can be located on the surface of the substructure or preferably on its end or side surfaces. The precise shape of the edge strip will depend on the hardness and elasticity of the plastic cover.